Retroviral vectors are a common tool for gene delivery (Miller, 1992, Nature 357: 455-460). The biology of retroviral proliferation enables such a use. Typically, wild type full length retroviral mRNA's serve both as a template for synthesis of viral proteins and as the viral genome. Such mRNA's encompass a region called the encapsidation signal which binds certain viral proteins thereby ensuring specific association of that mRNA with the produced virions. On infection of the target cell, reverse transcription of the retroviral mRNA into double stranded proviral DNA occurs. The retroviral enzyme, integrase, then binds to both long terminal repeats (LTR) which flank the proviral DNA and subsequently catalyzes the integration thereof into the genomic DNA of the target cell. Integrated proviral DNA serves as the template for generation of new full-length retroviral mRNA's.
Retroviral vectors have been tested and found to be suitable delivery vehicles for the stable introduction of a variety of genes of interest into the genomic DNA of a broad range of target cells. The ability of retroviral vectors to deliver unrearranged, single copy transgenes into cells makes retroviral vectors well suited for transferring genes into cells. Further, retroviruses enter host cells by the binding of retroviral envelope glycoproteins to specific cell surface receptors on the host cells. Consequently, the types of cells that a retrovirus can infect can be altered using pseudotyped retroviral vectors in which the encoded native envelope protein is replaced by a heterologous envelope protein that has a different cellular specificity than the native envelope protein (e.g., binds to a different cell-surface receptor as compared to the native envelope protein). The ability to direct the delivery of retroviral vectors encoding a transgene to a specific type of target cells is highly desirable for gene therapy applications.
The G glycoprotein from vesicular stomatitis virus (VSV-G) has been used extensively for pseudotyping retroviral vectors, including lentiviral vectors (see e.g., Emi et al., J. Virol. (1991) 65:1202-1207; Burns et al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037). VSV-G has been used in gene transfer protocols because of its broad species and tissue tropism and its ability to confer physical stability and high infectivity to vector particles (Yee et al, Methods Cell Biol. (1994) 43:99-112). However, the high fusogenicity of VSV-G causes rapid syncytia formation and cell death, making it difficult to generate stable cell lines expressing the protein (see e.g., Ory et al., Proc. Natl. Acad. Sci. USA (1996) 93:11400-11406). A number of groups have generated stable packaging and producer cell lines using VSV-G, but in all cases a repressible or inducible promoter regulates the expression of VSV-G (see e.g., Ory et al., Proc. Natl. Acad. Sci. USA (1996) 93:11400-11406; Farson et al., Hum. Gene Ther. (2001) 12:981-997; Kafri et al., J. Virol. (1999) 73:576-584). While regulated expression can potentially avoid the problem of VSV-G cytotoxicity, it makes stable production of viral vectors more complicated and impractical, and cell line generation more time-consuming.
Further, the broad tissue tropism of VSV-G can be disadvantageous in gene therapy applications because in some in vivo gene applications, it may be important to restrict the transfer and expression of a transgene to specific cell types. However, the broad distribution of the VSV-G receptor precludes this type of targeted transduction, even in instances of directed injection. VSV-G pseudotypes may transduce target cells of interest but following systemic administration may also transduce other cell types, thus, making it be problematic for use in gene therapy protocols. Antigen presenting cells (APC's), for instance, are cells that could cause deleterious immune responses if they are inadvertently transduced. Further, VSV-G pseudotyped lentiviral vectors can efficiently transduce APC's from mice and humans and can elicit immune responses against the transgene product thus negating the therapeutic effects of the expressed product (see e.g., Dyall et al, Blood (2001) 97:114-121; Chinnasamy et al, Hum. Gene Ther. (2000) 11:1901-1909; Metharom et al., Hum. Gene Ther. (2001) 12:2203-2213; VandenDriessche et al, Blood (2002) 100:813-822).
Systemic administration of viral vectors also exposes the vector particles to possible inactivation by serum complement (see e.g., Welsh et al., Nature (1975) 257:612-614). Complement inactivation of vectors is dependent on the species derivation of the cell line used to produce the vectors as well as the identity of the envelope glycoprotein used for pseudotyping (see e.g., Takeuchi et al., Nature (1996) 379:85-88; Takeuchi et al., J. Virol. (1 997) 71:6174-6178). VSV-G pseudotypes, regardless of producer cell type, will be inactivated by human serum complement while vectors pseudotyped with other envelope glycoproteins such as the MLV amphotropic envelope (Ampho) or RD114 are resistant to inactivation (DePolo et al., Mol. Ther. (2000) 2:218-222; Sandrin et al., Blood (2002) 100:823-832).
Thus, the use of VSV-G to pseudotype retroviruses for gene therapy applications has been attractive because of its high infectious titer and physical stability. In addition, VSV-G has been attractive due to its broad tropism, making it suitable for use with a wide variety of target cell types, including human cells. Unfortunately, this broad tropism can result in transduction of cells in which expression of the transgene is not desired, such as antigen presenting cells (APCs). However, in addition to drawbacks related to transduction of APCs, VSV-G, when constitutively expressed, can be cytotoxic, inhibit vector production, and render the vector sensitive to complement inactivation.
Consequently, because of the many disadvantages of VSV-G, there is considerable need for alternative envelope glycoproteins that have more suitable properties for use with retroviral vectors, including lentiviral vectors, in gene transfer applications. Of particular advantage would be the identification of envelope proteins that confer selective tropism, high titer in vivo, reduced cytoxicity, resistance to complement inactivation, and reduced ability to transduce APC's.